FORM 2
THE PATENT ACT 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION (Section 10 and Rule 13)
“PROCESS FOR THE PRODUCTION OF LACTIPLANTIBACILLUS
PLANTARUM NCIM5777”
TROPILITE FOODS PRIVATE LIMITED
DAVAR CAMPUS, TANSEN ROAD INDUSTRIES ESTATE, GWALIOR
MADHYA PRADESH – 474 002, INDIA
The following specification particularly describes the invention and the manner in which it is to be performed.

Field of the Invention
The invention relates to an optimized, economic process for the production of an isolated bacterial strain of Lactiplantibacillus plantarum having accession number NCIM5777, wherein the said strain being deposited at the National Collection of Industrial Microorganisms (NCIM), Pune, India, a national facility and microbial culture repository dedicated to isolation, preservation and distribution of authentic and industrially important microbial strains, and a member of World Federation for Culture Collections (WFCC/WDCM with Reg. No. 3).
Background of the Invention
Lactiplantibacillus plantarum (previously Lactobacillus plantarum) is a widespread member of the genus Lactiplantibacillus and commonly found in many fermented food products as well as anaerobic plant matter. Lactiplantibacillus plantarum is a kind of Lactic Acid Bacteria (LAB) which ferments arabinose, sucrose, glucose, fructose, galactose, maltose, dextran and the like, to generate lactic acids. In general, Lactiplantibacillus plantarum distributes widely in nature and is often isolated from dairy products (milk, cheese, and butter), kefir, fermented crops, kimchi, soil and the like.
Lactiplantibacillus plantarum has a feature to resist gastric acids and bile acids more strongly than other bacteria in a human body. Besides, it is reported as a beneficial bacterium to increase the secretion of cytokines in human immunocytes and decrease inflammation inducing factors, thereby helping to alleviate irritable bowel syndrome and improve atopic and allergic dermatitis. Lactiplantibacillus plantarum genome is also elucidated to generate an antibiotic substance referred to as lactolin that is effective to suppress herpes virus.
Lactiplantibacillus plantarum has significant antioxidant activities and helps to maintain intestinal permeability. It can be used as a probiotics microorganism and a component of health supplement. Lactiplantibacillus plantarum also maintains microflora balance and stabilization of digestive enzyme patterns in the gut.

Lactiplantibacillus plantarum NCIM5777 strain has been isolated from soil, and deposited at the National Collection of Industrial Microorganisms (NCIM), Pune, India. Lactiplantibacillus plantarum NCIM5777 has been identified as a potent probiotic culture which exhibits a good pH and temperature tolerance, stability with optimum pH 5 and 37°C. Thus, Lactiplantibacillus plantarum NCIM5777 has been selected for further investigations on process optimization of high cell density cultivation of probiotic culture.
Summary of the Invention
The present invention relates to an optimized, economic process for high cell density cultivation of an isolated bacterial strain of Lactiplantibacillus plantarum having accession number NCIM5777.
Accordingly, the present invention provides one or more of the following embodiments:
(1) A process for high cell density cultivation of Lactiplantibacillus plantarum
having accession number NCIM5777,
wherein, the said process comprises growing Lactiplantibacillus plantarum NCIM5777 in a production medium under optimized conditions,
wherein, the optimized conditions are the parameters / factors selected from, but not limited, to carbon source, nitrogen source, surfactant, pH, temperature, agitation rate or incubation period;
wherein, each parameter is optimized individually and incorporated in the experimental setup prior to optimization of the next factor till the final set of conditions and composition of the production medium is obtained.
(2) The process according to (1), wherein different physiological and
nutritional parameters are optimized by “one-variable-at-a-time” approach for
maximum cultivation of Lactiplantibacillus plantarum NCIM5777.

(3) The process according to (1), wherein optimal dry cell weight of Lactiplantibacillus plantarum NCIM5777 is obtained when carbon source is sucrose at a concentration 0.8%.
(4) The process according to (1), wherein optimal dry cell weight of Lactiplantibacillus plantarum NCIM5777 is obtained when nitrogen source is sodium
nitrate at a concentration of 0.8 %.
(5) The process according to (1), wherein Lactiplantibacillus plantarum grows
in pH range of 3.0-11.0, wherein optimal dry cell weight (DCW) of
Lactiplantibacillus plantarum NCIM5777 is obtained at pH 5.0
(6) The process according to (1), wherein the optimal mass of
Lactiplantibacillus plantarum NCIM5777 is observed in the temperature range of 20-
35°C, preferably at 30°C.
(7) The process according to (1), wherein Tween-80 enhances the cell biomass production of Lactiplantibacillus plantarum NCIM5777 at a concentration of 0.3%.
(8) The process according to (1), wherein incubation period of 24 hours yields
maximum biomass of Lactiplantibacillus plantarum NCIM5777.
(9) The process according to (1), wherein agitation rate of 150 rpm provides
enhances the cell biomass production of Lactiplantibacillus plantarum NCIM5777.
(10) A process for the high cell density cultivation of Lactiplantibacillus
plantarum NCIM5777, wherein the said process comprises, growing the
Lactiplantibacillus plantarum NCIM5777 in a fermentor with one of more of
optimized parameters selected from:
• carbon source (about 0.8%),
• Magnesium sulphate (0.08%),
• Surfactant (0.3%),
• inoculum density (4.0%) ,
• pH 5, temperature 30°C,
• 150 rpm (equivalent) agitation rate, and
• 24 hours incubation period.

Brief Description of the Drawings
Figure 1: Effect of the initial pH of the medium on cultivation of Lactiplantibacillus plantarum NCIM5777.
Figure 2: Effect of temperature on high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 after 24 hours of incubation.
Figure 3: Optimization of inoculum density for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777.
Figure 4: Optimization of agitation rates for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 at 30°C, in 24 hours.
Figure 5: Effect of different carbon sources on high cell density cultivation of Lactiplantibacillus plantarum NCIM5777.
Figure 6: Optimization of sucrose concentrations for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777.
Figure 7: Effect of nitrogen sources on high cell density cultivation of Lactiplantibacillus plantarum NCIM5777.
Figure 8: Optimization of NaNO3 concentration for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777.
Figure 9: Optimization of MgSO4 concentration for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777.
Figure 10: Effect of different surfactants on high cell density cultivation of Lactiplantibacillus plantarum NCIM5777.
Figure 11: Optimization of incubation period for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 under optimized conditions.

Detailed Description of the Invention
The present invention relates to an optimized, economic process for high cell density cultivation of an isolated bacterial strain of Lactiplantibacillus plantarum having accession number NCIM5777.
In one aspect, the present invention provides a process for the high cell density cultivation of Lactiplantibacillus plantarum NCIM5777, wherein the said process comprises, growing the Lactiplantibacillus plantarum NCIM5777 in a production medium under optimised conditions.
In one embodiment, Lactiplantibacillus plantarum NCIM5777 production is carried out using submerged fermentation (SmF), wherein the Lactiplantibacillus plantarum NCIM5777 was grown in liquid medium (broth) under shaking conditions.
In another aspect, the present invention provides a process for cultivation of probiotic strain of Lactiplantibacillus plantarum NCIM5777, wherein the process is optimised by one variable at a time approach for a maximum Lactiplantibacillus plantarum NCIM5777 cultivation.
In one embodiment, different physiological and nutritional parameters are optimized by one variable at a time approach for a maximum Lactiplantibacillus plantarum NCIM5777 cultivation. The optimized parameter is incorporated in the optimization of next parameter. This sequence of incorporation of optimized parameter is followed till a final set of optimized parameters are obtained.
In another embodiment, the physiological parameters include one or more of pH of the medium, temperature, inoculum density, agitation rate, and incubation period.
In yet another embodiment, the nutritional parameters include one or more of carbon source and concentration, nitrogen source and concentration, metal ion and its concentration, and surfactants and its concentration.
In yet another aspect, the present invention provides a process for the high cell density cultivation of Lactiplantibacillus plantarum NCIM5777, wherein the said process comprises, growing the Lactiplantibacillus plantarum NCIM5777 in a

fermentor having one of more of optimised parameters selected from about 0.8 % carbon source, about 0.8 % nitrogen source, 0.08% magnesium sulphate (MgSO4), 0.3% surfactant, 4.0% inoculum density, pH 5, temperature 30°C, 150 rpm (equivalent) agitation rate, and 24 hours incubation period.
In one embodiment, the preferred carbon source is Sucrose, the preferred nitrogen source is Sodium nitrate, and the preferred surfactant is Tween-80.
The present invention further include one or more embodiment, for example, in one embodiment, there is provided a process for high cell density cultivation of Lactiplantibacillus plantarum having accession number NCIM5777, wherein, the said process comprises growing Lactiplantibacillus plantarum NCIM5777 in a production medium under optimized conditions, wherein, the optimized conditions are the parameters / factors selected from, but not limited, to carbon source, nitrogen source, surfactant, pH, temperature, agitation rate or incubation period; wherein, each parameter is optimized individually and incorporated in the experimental setup prior to optimization of the next factor till the final set of conditions and composition of the production medium is obtained.
In another embodiment, there is provided a process for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777, wherein the said process comprises, growing the Lactiplantibacillus plantarum NCIM5777 in a fermentor with one of more of optimized parameters selected from: carbon source (about 0.8%), magnesium sulphate (0.08%), surfactant (0.3%), inoculum density (4.0%), pH5, temperature 30°C, agitation rate 150 rpm), and 24 hours incubation period.
In the following section, aspects are described by way of examples to illustrate the process of the invention. However, these do not limit the scope of the present invention. Several variants of these examples would be evident to persons ordinarily skilled in the art.
Experimental
Initial experiments for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 production showed that, the species of Lactiplantibacillus

plantarum NCIM5777 produces 6.1 mg DCW/ml of cell mass in 24 hours of incubation at 30°C in the unoptimized production medium containing Proteose peptone (2.0%), sodium nitrate (NaNO3) (0.8%), glucose (1.0%), Potassium dihydrogen phosphate (KH2PO4) (1.52 g/l), Potassium chloride (KCl) (0.52 g/l), Magnesium sulphate heptahydrate (MgSO4.7H2O) (0.52 g/l), Zinc sulphate heptahydrate (ZnSO4.7H2O), Ferrous sulphate heptahydrate (FeSO4.7H2O) and Cupric nitrate trihydrate (Cu(NO3)2.3H2O) (in traces), pH 7.0.
Taking these conditions as initial unoptimized conditions or control, optimization was carried out for enhancing high cell density of Lactiplantibacillus plantarum NCIM5777.
High cell density cultivation of Lactiplantibacillus plantarum NCIM5777 was carried out in 250 ml Erlenmeyer flasks containing 50 ml of the production medium (composition given above).
The following experiments were carried out: a) Selection of the production medium for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777; b) Process optimization for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 by one variable at a time (OVT) approach.
All experiments were carried out in triplicates and the standard deviation was calculated.
Selection of the production medium for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777
Inoculum preparation:
A loop full culture of Lactiplantibacillus plantarum was inoculated in bacterial minimal medium composed of Proteose peptone (10 g/l,) HM Peptone B (10g/l), Yeast extract (5 g/l), Dextrose (Glucose) (20 g/l), Tween 80 (Polysorbate 80) (1 g/l), Ammonium citrate (2 g/l), Sodium acetate (5 g/l), Magnesium sulphate (0.1 g/l), Manganese sulphate (0.05 g/l), supplemented with Dipotassium hydrogen phosphate (2.0 g/l ) (pH 7.0).

Production medium:
Here, eight different media designated from M1 to M8 where M1 is control were evaluated for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777. Composition of each of these medium is given in the below table.

Component Medi
M1
10
20
5 6 5 0.52
2
1.52
2
0.52
0.5
0.1
0.1
0.1 a desig M2 10
40 2
0.2
0.2
0.05 0.05 nated from M1 to M8

M3 M4 M5 M6
20
10
5
5
0.5
0.4
0.04 M7
10
10
10 5
0.02 M8 20
5
20 10
4
0.4
0.4
4 0.2
Glucose

10

Molasses

20 20

Sucrose

20

Tryptone

HM Peptone B

10 20

Soya Peptone

Yeast extract

20 10 10

NaNO3

sodium acetate

5 10

MgSO4. 7H2O

0.2 0.2 0.4

Ammonium acetate

Ammonium citrate

KH2PO4

0.2 0.2 0.4

KCl

FeCl3

0.1

MnSO4.4H2O

0.2 0.2

FeSO4.7H2O

ZnSO4.7H2O

Cu (NO3)2.3H2O

Table 1: Different production medium used for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777.
An inoculum level grown in nutrient broth with an OD600 (optical density of a sample measured at a wavelength of 600 nm) of 0.6–0.8) of 2% was used, and incubation was carried out at 30°C and 200 rpm for 24 hours. Samples were withdrawn at regular 6 hour intervals and were centrifuged at 10,000 rpm for 10 minutes in a refrigerated centrifuge at 4°C.

The palate was the culture source and was assayed for Dry Cell Weight (DCW) activity. The observations and evaluation of different production medium for high cell density cultivation of Lactiplantibacillus plantarum production after 24 hours of incubation are presented in Table 2.

S. No Production medium Lactiplantibacillus plantarum NCIM5777 (mg DCW/ml) Billion (CFU/g) Lactic acid (g/l)
1. M1 (control) 6.10± 0.13 288.00± 15 13.0± 0.1
2. M2 3.10± 0.12 49.00± 5 8.0± 0.1
3. M3 4.00± 0.08 142.00± 2 9.0.0± 0.1
4. M4 4.80± 0.09 52.00± 2 11.0.0± 0.2
5. M5 3.60± 0.15 132.00 ± 1 7.0± 0.1
6. M6 3.80± 0.09 112.00 ± 1 9.0± 0.1
7 M7 4.50±0.05 27.00± 2 11.0± 0.2
8 M8 5.00± 0.04 4.50±1 10.0±0.1
Table 2: Evaluation of different production mediums for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 after 24 hours of incubation
Table 2 shows that, the medium M1 which is also control supported maximum production (6.10± 0.13 mg DCW/ml) of cell cultivation of Lactiplantibacillus plantarum NCIM5777. The other media tested showed lesser yield. Thus, the medium M1 was selected as the best medium for further optimization studies for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777.
b) Process optimization for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 by one variable at a time (OVT) approach
After selecting the optimal medium for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777, process optimization was carried out by the one variable at a time approach.
Each parameter is optimized individually and incorporated in the experimental setup prior to optimization of the next factor till the final set of conditions and composition of the production medium is obtained. All these experiments were

carried out in triplicates and the standard deviation was calculated. The following parameters were optimized.
Optimization of pH:
pH is the most significant factor affecting growth and production of any micro organisms. Therefore, in this experiment, initial pH of the production medium was varied in a range from 3.0 to 12.0 (pH was adjusted using 1N NaOH or 1N HCl) and its effect on the production of Lactiplantibacillus plantarum NCIM5777 was carried out. Production medium supplemented with 2% Proteose peptone was inoculated with an inoculum level of 2% (OD600 of 0.6–0.8) and incubated at 30°C, 200 rpm for 24 hours. Samples were withdrawn and analyzed for mg DCW/ml of Lactiplantibacillus plantarum NCIM5777 and lactic acid activity and the observations are presented in the Figure 1.
Figure 1 clearly shows that Lactiplantibacillus plantarum grow in a wide pH range of 3.0-11.0 with higher biomass yields in acidic range. Optimal Dry Cell Weight (DCW) of Lactiplantibacillus plantarum NCIM5777 of 9.51 mg DCW/ ml was obtained at pH 5.0 as against 6.10 mg DCW/ml obtained with pH 7.0 (control) within 24 hours. Lactiplantibacillus plantarum NCIM5777 can also grow at the highly alkaline pH 10.0 (2.16 mg DCW/ml) and 11.0 (4.2 mg DCW/ml).
Optimization of Temperature:
Production of Lactiplantibacillus plantarum NCIM5777 was evaluated at different temperatures in the range from 20 to 50˚C under optimized conditions obtained so far. The Dry Cell Weight (DCW) of Lactiplantibacillus plantarum NCIM5777 and lactic acid concentration were estimated after 24 hours of incubation and observations are presented in Figure 2.
Figure 2, clearly shows that significant cell mass (DCW) of Lactiplantibacillus plantarum NCIM5777 was observed in the temperature range of 20-35°C with optimal production of 9.18 mg DCW/ml at 30°C. A significant loss in DCW was observed when incubation was carried out at temperatures higher than 35°C with minimum DCW cultivation titres of only 1.31 mg/ml obtained at 40°C.
Optimization of Inoculum density:

The effect of different inoculum densities ranging from 1.0% to 5.0% was examined on production of Lactiplantibacillus plantarum NCIM5777. The production was carried out at the optimized temperature and pH conditions in the medium containing 2.0% proteose peptone at pH 5.0, 30°C and 200 rpm for 24 hours. Observations are presented in Table 3 and Figure 3.

S. No Inoculum density (%) Lactiplantibacillus plantarum NCIM5777 (mg DCW/ml) Lactic acid (g/l)
1. 0.5 4.39± 0.15 1.5 ± 5
2. 1.0 6.24± 0.17 1.95 ± 5
3. 2.5 8.26± 0.20 2.75 ± 07
4. 2.0 9.23 ± 0.25 3.34 ± 95
5. 3.0 10.26 ±0.25 3.88 ± 10
6. 4.0 11.12 ± 0.30 4.21 ±0.12
7. 5.0 8.65± 0.21 3.701 ± 0.15
Table 3: Effect of inoculum density on high cell density cultivation of
Lactiplantibacillus plantarum NCIM5777
Table 3 and Figure 3 show that a yield of 11.12 mg DCW/ml of
Lactiplantibacillus plantarum NCIM5777 was obtained at an inoculum density of 4%
as against 9.23 mg DCW/ml obtained with 2.0% (control) within 24 hours. This
increase in DCW production of Lactiplantibacillus plantarum NCIM5777 is
approximately 1.20 fold higher.
Optimization of Agitation rate:
Agitation is an indirect measure of dissolved oxygen (DO) in the medium which affect the yield of cell mass/bi oa cti ve molecule. Hence, in order to evaluate the effect of agitation rate on the DCW production of Lactiplantibacillus plantarum NCIM5777, experiments were planned wherein the inoculated flasks were incubated at different agitation rates ranging from 0.0 (static) to 300 rpm at intervals of 50 rpm (Multitron, Switzerland). All other parameters (temperature, pH, inoculum density) were kept at their optimized levels. The Dry Cell Weight (DCW) of Lactiplantibacillus plantarum NCIM5777 and lactic acid concentration were

S. No Agitation rate (rpm) Lactiplantibacillus plantarum NCIM5777 (mg DCW/ml) Lactic acid (g/l)
1. Static 3.16± 0.15 1.49 ± 5
2. 50 6.20± 0.17 2.48 ± 5
3. 100 10.26± 0.20 3.10 ± 9
4. 150 14.23 ± 0.25 5.04 ± 15
5. 200 (control) 11.10 ± 0.30 4.28±12
6. 250 6.43 ± 0.25 2.06±5
7. 300 4.21± 0.21 1.87 ± 5
Table 4: Effect of different agitation rates on high cell density cultivation of
Lactiplantibacillus plantarum NCIM5777
Table 4 and F ig u re 4 s hows that opti mum cell mass of Lactiplantibacillus
plantarum NCIM5777 (14.23 mg DCW/ml) was achieved at 150 rpm as against 11.10
mg DCW/ml obtained at 200 rpm (control). Any change in agitation rate above or
below resulted in less biomass production with only 3.16 mg mg DCW/ml obtained
under static conditions. This increase in DCW production of Lactiplantibacillus
plantarum NCIM5777 is approximately 1.28 fold higher.
Effect of Carbon source:
The production medium selected for high cell density cultivation from this
species of Lactiplantibacillus plantarum NCIM5777 contains glucose as carbon
source at a concentration of 1.0% w/v. In order to evaluate the effect of different carbon sources on high cell mass cultivation of Lactiplantibacillus plantarum
NCIM5777, different monosaccharides, disacc ha rides, polysaccharides and sugar alcohols were individually added to the production medium at a concentration of 1.0% w/v. A negative control experiment devoid of any carbon source was also set up. After inoculation, incubation was carried out under parameters optimized so far (temperature, pH, inoculum density, agitation rate).

The Dry Cell Weight (DCW) of Lactiplantibacillus plantarum NCIM5777 and lactic acid concentration were estimated after 24 hours of incubation and observations are presented in Table 5 and Figure 5.

S. No Carbon sources Lactiplantibacillus plantarum NCIM5777 (mg DCW/ml) Lactic acid (g/l)
1 No carbon (negative control) 6.26 ± 0.20 2.53±0.10
2 Glucose (control) 14.23 ± 0.25 5.12± 0.15
3 Fructose 13.41 ± 0.30 3.12±0.10
4 Galactose 11.43 ± 0.20 5.14±0.20
5 Maltose 9.19 ± 0.10 3.54±0.10
6 Lactose 11.41 ±0.10 4.26±0.10
7 Sucrose 19.51 ± 0.40 6.48±0.10
8 Starch 9.29 ± 0.20 5.37±0.10
9 Mannitol 8.98 ± 0.10 3.51±0.05
10 Dextran 10.16 ± 0.25 3.25±0.10
11 Xylose 11.22 ± 0.2 4.14±0.10
12 Glycerol 10.61 ± 0.3 4.36±0.10
13 Rhamnose 8.19 ± 0.10 2.64±0.05
14 Arabinose 10.24 ± 0.10 3.32±0.1
15 Mannose 8.43 ± 0.10 0.81±0.09
16 Cellobiose 8.78 ± 0.10 4.26±0.15
17 Carboxymethylcellulose (CMC) 10.23 ± 0.30 1.65±0.1
18 Sorbitol 11.56 ± 0.20 2.64±0.1
Table 5: Effect of carbon source on high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 production
Table 5 and Figure 5 shows that sucrose supported significant higher titres of cell density of Lactiplantibacillus plantarum NCIM5777 (19.51 mg DCW/ml) as against glucose (14.23 mg DCW/ml control). Approximately 27.06% increase in high cell density was observed only when sucrose was used in place of glucose whereas other carbon sources supported cell mass production in the range of 8.0–11.0 mg DCW/ml of Lactiplantibacillus plantarum NCIM5777 respectively. Other than sucrose, most other sugars tested showed any significant increase in cell mass production of Lactiplantibacillus plantarum NCIM5777.

Thus, from the results it was evident that sucrose serves as better carbon source for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 and henceforth its concentration was further optimized for maximum cultivation of Lactiplantibacillus plantarum NCIM5777.
Optimization of Sucrose concentration:
After confirming sucrose as the best carbon source, its concentration was optimized for high cell density cultivation. Experimentally, different concentrations of sucrose (0.2 to 2.0 % w/v) were added to the production medium under conditions optimized so far (temperature, pH, inoculum density, agitation rate).
The Dry Cell Weight (DCW) of Lactiplantibacillus plantarum NCIM5777 and lactic acid concentration were estimated after 24 hours of incubation and observations are presented in Figure 6.
Figure 6 clearly shows that cell mass of Lactiplantibacillus plantarum NCIM5777 slightly increased to 21.39 mg DCW/ml on decreasing sucrose concentration to 0.8%. However, any further increase or decrease in sucrose concentration led to a decline in the yield of cell mass of Lactiplantibacillus plantarum NCIM5777.
Effect of Nitrogen source:
In order to determine the best nitrogen source for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777, sodium nitrate (0.6% w/v) and proteose peptone (2.0% w/v) was replaced with different inorganic and organic nitrogen sources in the production medium. All the nitrogen sources used had % equivalent nitrogen similar to that of 0.6% (w/v) sodium nitrate and 2% proteose peptone. High cell density cultivation of Lactiplantibacillus plantarum NCIM5777 was carried out under the optimized conditions so far (temperature, pH, inoculum density, agitation rate).
The Dry Cell Weight (DCW) of Lactiplantibacillus plantarum NCIM5777 and lactic acid concentration were estimated after 24 hours of incubation and observations are presented in Figure 7.

Figure 7 clearly shows that there is no enhancement in cell mass production of Lactiplantibacillus plantarum was observed on replacement of control nitrogen with other nitrogen sources in the production medium. Amongst the inorganic nitrogen sources, only sodium nitrate supported good cell density cultivation, while others showed low titres of cell mass. However, very low amount of DCW was observed when corn steep liquor (CSL), casein hydrolysate and arginine were used.
Optimization of Sodium nitrate concentration:
After selecting Sodium nitrate (NaNO3) as the nitrogen source, various concentrations in the range 0.2–1.2 % were evaluated for high cell density cultivation of Lactiplantibacillus plantarum. All other parameters used were at their optimized values as described above.
The Dry Cell Weight (DCW) of Lactiplantibacillus plantarum NCIM5777 and lactic acid concentration were estimated after 24 hours of incubation and observations are presented in Table 6 and Figure 8.

S. No Concentration of NaNO3 (%) Lactiplantibacillus plantarum NCIM5777 (mg DCW/ml) Lactic acid (g/l)
1. 0.2 14.19 ± 0.25 4.87 ± 0.10
2. 0.4 17.76 ± 0.30 5.27 ± 0.085
3. 0.6 (control) 21.42 ± 0.50 6.68 ± 0.10
4. 0.8 24.38 ± 0.50 7.86 ± 0.15
5. 1.0 16.92 ± 0.40 6.41 ± 0.10
6. 1.2 15.37 ± 0.30 5.29 ± 0.12
Table 6: Effect of NaNO3 concentration on cell mass production of
Lactiplantibacillus plantarum NCIM5777
Table 6 and Figure 8 clearly shows that 24.38 mg DCW/ml of Lactiplantibacillus plantarum was produced at 0.8 % NaNO3 concentration as compared to 0.6% of NaNO3 which serve as control. On increasing the concentration of NaNO3 to 1.0 %, significant lesser DCW of Lactiplantibacillus plantarum

NCIM5777 was observed. Any further change in concentration leads to a decline in cell density yields.
Effect of Metal ions:
In the high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 medium (M1), magnesium sulphate (MgSO4) as Mg2+ and KH2PO4 as potassium (K+) are present at a concentration of 0.05 and 0.15%. Hence, to evaluate the effect of different metal ions, experiments were designed wherein; MgSO4 and KH2PO4 were replaced with different metal ions at a concentration of 0.2% equivalent to the total sum of MgSO4 and KH2PO4 along with negative and positive control for 24 hours.
Experimentally, three set of production medium were prepared.
Set I: M1 medium optimized so far (as described above)
Set II: Optimized medium M1 devoid of MgSO4 and KH2PO4 (-ve control)
Set III: Optimized M1 medium individually supplemented with different ions viz. Ca2+, Fe2+, Pb2+, Mn2+, Zn2+, Co2+, Ag+, Hg2+, Cu2+, Mg2+ and K+ at a concentration of
0.2%.
Inoculation and incubation was carried out under conditions optimized so far. The Dry Cell Weight (DCW) of Lactiplantibacillus plantarum NCIM5777 and lactic acid concentration were estimated and observations are presented in Table 6.

S. No Metal ions Lactiplantibacillus plantarum NCIM5777 (mg DCW/ml) Lactic acid (g/l)
1. Control (optimized M1 medium) 24.38±0.50 7.86±0.15
2. No metal salts 14.32 ± 0.25 3.77±0.10
3. MgSO4 (Mg2+) 21.29 ± 0.25 7.91±0.15
4. CaCl2 (Ca2+) 13.63 ± 0.30 4.52±0.20
5. KH2PO4 (K+) 20.73 ± 0.30 5.32±0.12
6. FeCl3.6H2O (Fe2+) 15.54 ± 0.28 4.69±0.15
7. CuCl2.2H2O (Cu2+) 14.63 ± 0.25 5.16±0.095
8. ZnCl2 (Zn2+) 16.34 ± 0.32 3.79±0.15

9. COCl2.6H2O (Co2+) 13.68 ± 0.30 5.22±0.10
10. AgCl (Ag+) 12.54 ± 0.15 4.31±0.075
11. HgCl2 (Hg2+) 9.42 ± 0.10 5.54±0.20
12. MnCl2 (Mn2+) 11.22 ± 0.25 5.21±0.25
13. PbCl2 (Pb2+) 9.49 ± 0.22 3.32±0.15
Table 7: Effect of different metal ions on high cell density cultivation of
Lactiplantibacillus plantarum NCIM5777
Table 7 clearly shows that metal ions were important for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777. However, if only K+ is added, 20.73 mg DCW/ml and if Mg2+ is added in the production medium 21.29 mg DCW/ml of Lactiplantibacillus plantarum NCIM5777 is produced. Very low cell yield (14.32 mg DCW/ml) was obtained in medium devoid of any metal salts. Similarly, mercury (Hg+), silver (Ag+), lead (Pb2+) and zinc (Zn2+) were inhibitory for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777. On the other hand, when both metal ions, i.e., Mg2+ and K+ present in the optimized M1 medium obtained so far were used, 24.38 mg DCW/ml is obtained. Thus, it is necessary to optimize concentration of Mg2+ and K+ ions, so that an enhanced amount of DCW is produced. Therefore, the optimized production medium obtained so far was used for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 without any additional metal ions.
Optimization of MgSO4 concentration:
The effect of different concentrations of Mg2+ ions in the range of 0.02-0.1% were examined on high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 in the optimized medium containing KH2PO4 at a concentration of 0.15%.
The Dry Cell Weight (DCW) of Lactiplantibacillus plantarum NCIM5777 and lactic acid concentration were estimated and observations are presented in Table 8 and Figure 9.

S. No Concentration of MgSO4 (%) Lactiplantibacillus plantarum NCIM5777 Lactic acid (mg/ml)

(mg DCW/ml)
1. 0.02 15.1 ± 0.25 5.35±0.10
2. 0.04 18.89 ± 0.32 6.27±0.085
3. 0.05 (control) 24.32 ± 0.54 7.81±0.15
4. 0.06 26.23 ± 0.51 9.02±0.15
5. 0.08 29.21 ± 0.53 10.32±0.15
6. 0.10 25.69 ± 0.48 8.29±0.22
Table 8: Optimization of MgSO4 concentration for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 production
Table 8 and Figure 9 clearly show that maximum production of 29.21 mg DCW/ml was obtained when Mg2+ ions at concentration of 0.08% was present. Any increase or decrease in the concentration of Mg2+ ions resulted in the decline of cell density of Lactiplantibacillus plantarum NCIM5777 production. Now, the optimized medium for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 gives 29.21 mg DCW/ml of Lactiplantibacillus plantarum NCIM5777 which contains proteose peptone (2.0%), NaNO3 (0.8%), sucrose (0.8%), KH2PO4 (1.52 g/l), KCl (0.52 g/l), MgSO4.7H2O (0.8 g/l), FeSO4.7H2O, ZnSO4.7H2O and Cu(NO3)2.3H2O (in traces); pH 5.0, 30°C, 150 rpm and inoculum density of 4.0%. In this optimized medium, different concentrations of K+ ions were evaluated along with the optimized concentration of MgSO4 i.e. 0.08%.
Optimization of KH2PO4 concentration:
To find the optimum concentration of KH2PO4 for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777, experiments were executed in the medium optimized so far containing KH2PO4 in the range of 0.05 to 0.25%. Culture were raised for 24 hours and analyzed for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777.
The Dry Cell Weight (DCW) of Lactiplantibacillus plantarum NCIM5777 and lactic acid concentration were estimated and observations are presented in Table 8.

S. No Concentration
of KH2PO4 (%) Lactiplantibacillus plantarum NCIM5777 (mg DCW/ml) Lactic acid (g/l)

1. 2. 3. 4. 5. 0.05 17.19 ± 0.26 5.35±0.10

0.10 24.69 ± 0.30 6.27±0.085

0.15 (control) 29.21 ± 0.42 10.32±0.15

0.20 25.33 ± 0.40 9.02±0.15

0.25 20.29 ± 0.52 8.91±0.15
Table 9: Optimization of KH2PO4 concentration for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777
Table 9 clearly shows that significant biomass was produced in the potassium concentration range of 0.1-0.2% with an optimal 29.21 mg DCW/ml at 0.15% (control). A significant loss in biomass was observed when incubation was carried out at higher or lower K+ concentration as against control. At 0.05% of K+ concentration a minimum biomass of L Lactiplantibacillus plantarum NCIM5777 of 17.19 mg DCW/ml was obtained.
Effect of trace element:
The medium contains ZnSO4, FeSO4 and Cu(NO3)2 as trace elements. Since trace element play a pivotal role in cell mass production, the effect of these trace elements on high biomass production were evaluated.
Experimentally, three set of production medium were prepared.
Set 1: Optimized production medium M1 (control)
Set 2: Optimized Medium M1 devoid of ZnSO4, FeSO4 and Cu (NO3)2 (-ve control)
Set 3: Optimized production medium M1 individually supplemented with different trace element viz. ZnSO4, FeSO4 and Cu(NO3)2 at a concentration of 1.0 mM.
The Dry Cell Weight (DCW) of Lactiplantibacillus plantarum NCIM5777 and lactic acid concentration were estimated and observations are presented in Table 10.

S. No Trace element Lactiplantibacillus plantarum NCIM5777 (mg DCW/ml) Lactic acid (mg/ml)
1. Control 29.21 ± 0.53 10.32±0.15
2. (-ve control) 22.69 ± 0.48 7.29±0.22
3. ZnSO4 24.1 ± 0.25 8.55±0.20
4. FeSO4 25.89 ± 0.32 8.97±0.25
5. Cu(NO3)2 27.29 ± 0.54 8.91±0.23
Table 10: Effect of trace elements on high cell density cultivation of Lactiplantibacillus plantarum NCIM5777
Table 10 clearly shows that trace element enhances cell density of Lactiplantibacillus plantarum NCIM5777 as it has been observed against –ve control wherein no trace element was present. In this condition, only 22.69 mg DCW/ml of Lactiplantibacillus plantarum NCIM5777 was produced. However, when all the three trace elements were present in the medium, high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 increases to 29.21 mg DCW/ml. Observations also showed that even though if Cu(NO3)2 (traces) was present solely, then also more or less same amount of cell density i.e., 27.29 mg DCW/ml was recorded. Thus, the optimized production medium obtained so far can be used for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 without any additional trace element.
Effect of Surfactants:
Surfactants is a significant factor for enhancing high cell density cultivation of cell mass, It is important to evaluate the effect of various surfactants on high cell density cultivation of Lactiplantibacillus plantarum NCIM5777. Experimentally, 0.2% of Tween 20, 40, 80, Triton X-100 and Saponin were supplemented individually to 50 ml of optimized production medium (in 250 ml Erlenmeyer flasks) and inoculated with 4.0% inoculum. The incubation was carried out at 30°C and 150 rpm. The observations for DCW of Lactiplantibacillus plantarum NCIM5777 and lactic acid after 24 hours were presented in Table 11 and Figure 10.

S. No Surfactants Lactiplantibacillus plantarum NCIM5777 (mg DCW/ml) Lactic acid (g/l)
1. No surfactant (control) 29.25 ± 0.53 10.25 ± 0.15
2. Tween-20 21.6±0.5 6.92±0.15
3. Tween-40 29.4±0.6 8.25±0.2
4. Tween-80 32.6±0.8 11.17±0.2
5. TritonX-100 21.4±0.5 7.30±0.2
6. Saponin 27.6±0.6 8.84±0.2
Table 11: Effect of different surfactants on high cell density cultivation of
Lactiplantibacillus plantarum NCIM5777 production
Table 11 and Figure 10 clearly show that addition of Tween-80 enhances the
cell biomass of Lactiplantibacillus plantarum NCIM5777 to 32.6 mg DCW/ml.
Therefore, Tween-80 was incorporated into the optimized medium and its concentration was subsequently optimized.
Optimization of Tween-80 concentration :
Tween-80 was supplemented to the production medium in concentrations ranging from 0.1 to 0.5%. After incubation under conditions optimized so far, samples were withdrawn after 24 hours of incubation and evaluated for cell density of
Lactiplantibacillus plantarum NCIM5777 (mg DCW/ml) and Lactic acid. The observations for DCW of Lactiplantibacillus plantarum NCIM5777 and lactic acid
concentration after 24 hours were presented in Table 12.

S. No Concentration (% v/v) Lactiplantibacillus plantarum NCIM5777 (mg DCW/ml) Lactic acid (g/l)
1. 0.1 31.09 ± 0.83 10.77 ± 0.2
2. 0.2 (Control) 32.58 ± 0.80 11.17 ± 0.2
3. 0.3 35.65 ± 0.50 11.21 ± 0.2
4. 0.4 32.7 ± 0.62 10.76 ± 0.2
5. 0.5 30.5 ± 0.53 9.18 ± 0.2

Table 12: Optimization of different concentrations of Tween-80 for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777
Table 12 clearly shows that on increasing Tween-80 concentration to 0.3%, optimal cell mass 35.65 mg DCW/ml were obtained which is 17.95% higher than the yield obtained in absence of any surfactant (29.25 mg DCW/ml).
Optimization of Incubation period:
Once physiological and nutritional factors were optimized, it is necessary and important to evaluate the time period for obtaining maximum cell density. Hence, an experiment was planned wherein biomass production of Lactiplantibacillus plantarum NCIM5777 was evaluated in relation to time under optimized condition obtained so far which include Proteose peptone (2.0%), sucrose (0.8%), sodium nitrate (0.8%), KH2PO4 (0.15%), MgSO4 (0.08%), KCl (0.52 g/l), Tween-80 (0.3%), ZnSO4.7H2O, FeSO4.7H2O and Cu(NO3)2.3H2O (traces), pH 5.0 and inoculated with 4.0% culture at 30°C, 150 rpm for 24 hours. The observations in terms of mg DCW/ml of Lactiplantibacillus plantarum NCIM5777, colony-forming unit (CFU) and concentration of lactic acid (g/l) are presented in the Figure 11.
Figure 11 clearly shows that, maximum biomass yield was obtained in 24 hours with no further increase on continued incubation. Therefore, 24 hours was the optimal incubation period for maximum biomass production of Lactiplantibacillus plantarum NCIM5777.
It is clear from the results of process optimization using “one-variable-at-a-time” approach, that both physiological and nutritional factors showed significant influence on high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 under submerged fermentation. The optimization of these factors resulted in the final yield of 35.65 mg DCW/ml of Lactiplantibacillus plantarum NCIM5777 in 24 hours. This yield is 5.84 fold higher as against the initial un-optimized medium wherein 6.1 mg DCW/ml of Lactiplantibacillus plantarum NCIM5777 was produced. The final optimized conditions obtained for high cell density cultivation of Lactiplantibacillus plantarum NCIM5777 after one variable at a time approach are summarized in Table 13.

Factors Optimized conditions
Carbon source and concentration Sucrose, 0.8 %
Nitrogen source and concentration NaNO3, 0.8 %
Magnesium sulphate (MgSO4) 0.08 %
Surfactant and concentration Tween-80, 0.3%
Inoculum density 4.0 %
Temperature 30°C
pH 5.0
Agitation rate 150 rpm
Incubation period 24 hours
Initial yield of high cell density cultivation of
Lactiplantibacillus plantarum NCIM5777
(Un-optimized condition) 6.1 mg DCW/ml
Final yield of high cell density cultivation of
Lactiplantibacillus plantarum NCIM5777
(After one variable at a time) 35.65 mg DCW/ml
Fold increase 5.84
Table 13: Optimized conditions (physiological and nutritional parameters) obtained by “one-variable-at-a-time” method for maximum high cell density cultivation of Lactiplantibacillus plantarum NCIM5777
While the present invention has been described in terms of its specific aspects, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

We Claim:
Claim 1: A process for high cell density cultivation of Lactiplantibacillus plantarum having accession number NCIM5777, wherein, the said process comprises growing Lactiplantibacillus plantarum NCIM5777 in a production medium under optimized conditions; wherein, the optimized conditions are the parameters selected from carbon source and concentration, nitrogen source and concentration, surfactant, pH, temperature, agitation rate or incubation period; wherein, each parameter is optimized individually and incorporated in the experimental setup prior to optimization of the next parameter ti ll the final set of conditions and composition of the production medium is obtained.
Claim 2: The process according to claim (1), wherein the optimized carbon source is sucrose at a concentration of 0.8%.
Caim 3: The process according to claim (1), wherein the optimized nitrogen source is sodium nitrate at a concentration of 0.8 %.
Claim 4: The process according to claim (1), wherein the optimized pH value is 5.0; the optimized temperature is 30°C.
Caim 5: The process according to claim (1), wherein optimized surfactant is Tween-80 at a concentration of 0.3%.
Claim 6: The process according to claim (1), wherein optimized incubation period is 24 hours.
Caim 7: The process according to claim (1), wherein a g itati on rate is 150 rpm .
Caim 8: A process for high cell density cultivation of Lactiplantibacillus plantarum
NCIM5777, wherein the said process comprises, growing the Lactiplantibacillus
plantarum NCIM5777 in a fermentor with one or more of optimized parameters selected from:
• Carbon source (about 0.8%),
• Nitrogen source (about 0.8%),
• Magnesium sulphate (0.08 %),
• Surfactant (0.3%),

• inoculum density (4.0%),
• pH 5, temperature 30°C,
• 150 rpm (equivalent) agitation rate, and
• 24 hours incubation period.
Claim 9: A process for high cell density cultivation of Lactiplantibacillus plantarum having accession number NC I M5777,
wherein, the said process comprises growing Lactiplantibacillus plantarum
NCIM5777 in a production medium under opti mi z e d c onditions as shown below:

Factors Optimized conditions
Carbon source and concentration Sucrose, 0.8 %
Nitrogen source and concentration NaNO3, 0.8 %
Magnesium sulphate (MgSO4) 0.08 %
Surfactant and concentration Tween-80, 0.3%
Inoculum density 4.0 %
Temperature 30°C
pH 5.0
Agitation rate 150 rpm
Incubation period 24 hours
Initial yield of high cell density cultivation of
Lactiplantibacillus plantarum NCIM5777
(Un-optimized condition) 6.1 mg DCW/ml
Final yield of high cell density cultivation of
Lactiplantibacillus plantarum NCIM5777
(After one variable at a time) 35.65 mg DCW/ml
Fold increase 5.84
wherein, each parameter is optimized individually and incorporated in the experimental setup prior to optimization of the next parameter till the final set of conditions and composition of the production medium is obtained.
(Claim 10) A process for the high cell density cultivation of Lactiplantibacillus plantarum having accession number NCIM5777, substantially as described herein and in the appended drawings.